0270~6474/83/0306-1279$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 3,, No. 6, pp. 1279-1288 Printed in U.S.A. June 1983

FAST AXONAL TRANSPORT IN PERMEABILIZED LOBSTER GIANT IS INHIBITED BY VANADATE’

DAVID S. FORMAN,*, 2 KURT J. BROWN,* AND DAVID R. LIVENGOODS

* Department of Anatomy, Uniformed Services University of the Health Sciences and $ Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20814

Received October 22, 1982; Revised January 17, 1983; Accepted January 18, 1983

Abstract We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5’-triphosphate (ATP)-free “internal” medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 PM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 PM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dynein- like molecule may play a role in the mechanism of fast axonal transport.

Fast axonal transport can be directly visualized in the mechanism of transport do not cross the axonal living axons by light microscopy (Smith, 1971,1977,1980; plasma membrane. One approach to circumvent this Kirkpatrick et al., 1972; Cooper and Smith, 1974; Forman problem that has been useful for studying other types of et al., 1977a, b; Forman and Shain, 1981; Allen et al., cell motility is to destroy the permeability barrier of the 1982a, b; Forman, 1982c; reviewed in Grafstein and For- plasma membrane with detergents in a medium that man, 1980). Microscopically visible organelles move in permits reactivation of the movement. Detergent-per- both the anterograde and retrograde directions by salta- meabilized cells have proved useful for analyzing the tory movements similar to those seen in a variety of mechanisms underlying other types of cell motility, such other cells (Rebhun, 1972; Forman, 1982b). The cellular as muscle contraction, ciliary motion, chromosome move- mechanism of fast transport is poorly understood. Many ment, and cytokinesis (Cande and Wolniak, 1978; Cande experimental probes that might be useful for studying et al., 1981). Recently, methods have been reported for reactivating saltatory movement in fibroblasts (Forman, 1981; 1982b), (Clark and Rosenbaum, ’ This work was supported by the Uniformed Services University of 1982; Stearns and Ochs, 1982) and neuroblastoma cells the Health Sciences, Protocols R07035 and R07032, and United States (Stearns and Boggs, 1981) permeabilized with detergent, Public Health Service Grant GM30450. The opinions or assertions and in isolated axoplasm (Brady et al., 1982). In contained herein are the private ones of the authors and are not to be this paper we describe a method for permeabilizing lob- construed as official or reflecting the views of the Department of ster axons with saponin and reactivating fast transport Defense or the Uniformed Services University of the Health Sciences. with exogenous ATP. We have used this method to study We greatly appreciate a loan of equipment from the Naval Medical the effects of vanadate, an inhibitor of dynein (the ATP- Research Institute, Bethesda, Maryland. We wish to thank Mark ase involved in the movement of cilia and flagella, re- Promersberger for excellent technical assistance, Dr. Mark Adelman viewed in Warner and Mitchell, 1980). Vanadate, which for valuable discussion, and Clayton Cisar and Ed Lundgren, Labora- cannot normally enter living axons, reversibly inhibits tory Animal Medicine Department, Uniformed Services University of the Health Sciences, for help in procuring lobsters. fast organelle transport in the permeabilized axons. This ‘To whom correspondence should be addressed at Department of finding suggests that a cytoplasmic dynein-like molecule Anatomy, Uniformed Services University of the Health Sciences, may play a role in the mechanism of fast axonal trans- School of Medicine, 4301 Jones Bridge Road, Bethesda, MD 20814. port.

1279 1280 Forman et al. Vol. 3, No. 6, June 1983

Materials and Methods was covered by a 45 x 45 mm mask (equivalent to an Isolation of giant axons. Lobsters (Homarus ameri- area of 100 mm’). Long saltations and “tugging move- canus) from commercial sources, weighing 0.5 to 1 kg, ments” (see “Results”) with net displacements of greater were maintained in aerated holding tanks in artificial sea than 1 pm were counted during successive 1-min periods water at 4°C for up to 3 days. Walking legs were removed of videotape record. Counts from four equally spaced immediately before dissection and placed in an regions (totaling 40% of the screen area) were added “external” medium containing 450 mM NaCl, 15 mu KCl, together to give a representative index of the frequency 25 mu CaCL, 4 mM MgS04, 4 mu MgC12, and 10 mu of organelle movements (see Forman, 1982a). Particle HEPES (sodium salt), pH 7.4. Motor nerves in the mer- velocities were measured from videotape records using a opodite (Wiersma, 1961) were exposed, and the proximal 610E Video Pointer (Colorado Video, Boulder, Colorado). and distal ends of 3- to 5-cm-long segments were tied Permeabilization. Organelle movements inside an with surgical threads of different diameters so that the axon were observed in external medium to provide a base direction of movement could later be identified. The line. The axon was then incubated for 10 min in an nerves were placed in 85-mm plastic Petri dishes contain- “internal” medium (modified from Abercrombie et al., ing about 15 ml of external medium and were anchored 1981) that contained 300 mM potassium aspartate, 20 mM by pushing the ends into a 5-mm-thick layer of sodium aspartate, 344 mM glycine, 10 mM MgCL, 5 mu Sylgard (Dow Corning) on the bottom. Single giant motor EGTA, and 10 mM HEPES, pH 7.3. The axon was then axons or adherent pairs were isolated using a Zeiss mi- permeabilized by incubation for 15 min in internal me- crosurgical dissecting microscope and darkfield illumi- dium plus 0.02% saponin (Sigma) and 1% dimethyl sulf- nation. In most experiments the axons studied were oxide (DMSO). The saponin was added to the medium either the “fast closer” or one of the two “opener” axons from a stock solution of 2% (w/v) saponin dissolved in to the dactylopodite (Wiersma, 1961; Kravitz et al., 1965); DMSO. DMSO alone (1%) did not permeabilize the axon there was no obvious difference in movement between or noticeably affect transport. The axon was then per- the different types of axons. The diameters of the axons fused with various reactivation media consisting of ad- ranged from 30 to 50 pm. ditional components (described under “Results”) dis- A coverslip longer than the nerve segment was slipped solved in internal medium without saponin or DMSO. underneath it, and the medium was gently removed with Results a syringe. The coverslip with the adhering axon was then quickly inverted and lowered onto a pool of medium on Fast axonal transport of microscopically visible or- a spacer slide. The spacer slide consisted of a 74 mm x ganelles in intact axons. With conventional Nomarski 50 mm glass slide to which two 60-mm long no. 1 cover- optics, numerous organelles can be seen moving inside slips were glued with Permabond 910 adhesive, leaving a living lobster giant axons. The sheath surrounding each gap of 10 mm between them. The sides of the coverslip isolated axon, consisting of several layers of thin glial were sealed with melted Kronig wax. Solutions were cells and collagen fibers, did not excessively interfere changed by pipetting fluid at one end of the coverslip and with good visualization of organelles in the axoplasm absorbing it at the other end with filter paper. (Fig. 1). The organelles and their movements resembled Video microscopy. The axons were observed with No- those that have been described in other types of axons marski differential interference contrast optics using a (Smith, 1971, 1972, 1977, 1980; Kirkpatrick et al., 1972; Zeiss basic microscope with a X 40 planachromatic ob- Cooper and Smith, 1974; Forman et al., 1977a, b; reviewed jective (numerical = 0.65). A Calflex heat re- in Grafstein and Forman, 1980; Forman, 1982b). Briefly, flection filter was used to protect the specimen, and a two classes of organelles could be distinguished: elon- Zeiss VG9 filter provided monochromatic (546 & 5 nm) gated mitochondria and small, round or ellipsoidal, vesic- light. Best results were obtained with the polarizer par- ular “particles” (Figs. 1 and 2, a to c).~ Mitochondria allel to the axon. The microscopic image was magnified varied in length from a few micrometers to longer than and recorded using a video microscopy system (Wil- 40 w and were usually aligned parallel to the long axis lingham and Pastan, 1978) consisting of a highly sensitive of the axon. Mitochondria were found distributed SIT video (RCA TC1030H), a time-lapse video- throughout the axoplasm but were especially concen- tape recorder (Panasonic NV-8030 or NV-8050), and a trated immediately below the axonal plasma membrane. monitor (RCA TC1209). Time was continuously dis- More particles than mitochondria were seen moving played on the video record by a date and time generator (Table I). The movement of both particles and mito- (RCA TC1440B). The axons were observed at a magni- chondria appeared to be similar to the saltatory organelle fication of x 4500 on the monitor screen and recorded in time-lapse mode so that time was compressed by a factor 3 By the use of the Allen video-enhanced contrast, differential inter- of 9. Temperature, which was monitored by a thermistor ference contrast (AVEC-DIC) technique, Allen et al. (1982a, b) have taped to the stage, ranged from 21’ to 24°C. reported detecting three classes of organelles: mitochondria, “medium” To measure the proportion of organelles moving in the size particles, and “small” particles. They propose that the small retrograde and anterograde directions, the numbers of particles, which move predominantly in the anterograde direction, particles crossing a vertical line at the center of the represent SO- to 50-nm vesicles that have not previously been observed by light microscopy (Allen et al., 1982a, b; Brady et al., 1982; Fahim et monitor screen were counted for 5-min segments of vi- al., 1982). In our study, like all previous studies using conventional deotape record. Elongated organelles, presumed to be microscopy, “small” vesicles were not detected, all particles described mitochondria, were counted separately. To measure the in this paper were of the “medium” type. The implications of this are frequency of organelle movements, the monitor screen considered further under “Discussion.” The Journal of Neuroscience Vanaclate Inhibits Fast Axonal Transport in Permeabilized Axons 1281

I). In both regions the proportion of mitochondria moving in each direction was similar to the proportion of particles moving in the same direction. When the external medium was replaced by internal medium before saponin treatment, there was no change in the velocities, frequency, or direction of particle move- ment. Movement continued for more than 1 br in internal medium without any obvious changes in organelle move- ment. Intracellular recordings in other axons confirmed that the axons were reversibly depolarized in internal medium, as would be expected from the high K+ concen- tration. These results are consistent with previous dem- onstrations that fast transport is not affected by mem- brane depolarization (see Grafstein and Forman, 1980). ATP-dependent fast transport of organelles in axons permeabilized with saponin. The permeabilization pro- cedure produced a reproducible series of effects on axonal structure and organelle movement. Between 5 and 10 min after the addition of the saponin, several changes occurred that indicated breakdown of the plasma mem- Figure 1. Low magnification view of isolated lobster giant brane permeability barrier. Some mitochondria devel- axon (bar = 10 pm). A sheath (s) surrounds the axon. The axon oped a series of constrictions and periodic swellings re- contains elongated mitochondria and small spherical or ellip- sembling a string of beads (Fig. 2d), whereas others soidal vesicles (“particles”). fragmented. Large empty vacuoles were rarely seen in normal axoplasm, but when such vacuoles were present movements seen in other types of cells (Rebhun, 1972). they lost refractility and lysed during the permeabiliza- The organelles moved in straight translocations parallel tion. Small particles did not show any obvious lysis or to the long axis of the axon, (Fig. 2, u to c), with velocities morphological change, but their saltatory movements usually between 0.3 and 2.4 pm/set (Table I). The move- decreased markedly. By 15 min after the arrival of the ment was often interrupted by pauses of variable dura- saponin, virtually all saltatory movement had ceased. tion or by brief reversals of direction. Each organelle Organelles remained immobile, without any Brownian moved predominantly in one “major” direction (Forman movement. No additional changes were seen if the axon et al., 1977a). was left in the saponin-containing medium for up to an Despite overall similarities, there were some interest- hour. ing differences between the patterns of organelle move- There was also no movement if the medium was re- ment that we observed in lobster axons and those that placed by saponin-free internal medium without ATP. have been described in other types of axons: (1) Although However, when the axon was perfused with internal many mitochondria in the lobster axon were stationary, medium containing 1 mM MgATP, saltatory organelle mitochondrial movement in these axons appeared to be movement dramatically reappeared (Figs. 2 and 4). The more frequent (Table I) than in vertebrate axons (Cooper reactivated movements generally resembled those seen and Smith, 1974; Forman et al., 1977a). (2) In both in intact axons. Although saltations tended to be shorter vertebrate (Cooper and Smith, 1974; Forman et al., 1977a, and slower than normal, some particles traversed the b; Smith 1971, 1972, 1980) and crustacean axons (Smith, entire field of view (40 m). Movement occurred in both 1977; Adams, 1982) the great majority of the particles the anterograde and retrograde directions. Most of the that are large enough to be seen with conventional optics moving organelles were particles, but mitochondrial sal- have been seen to move in the retrograde direction, and tations were also observed. The number of moving par- no regional differences in particle direction have been ticles was variable but was always less than in the intact reported. However, we found a regional difference in the axon (Fig. 4). The number of movements during the first lobster axons: in the interior of the axon more than 80% 5 min of reactivation with 1 mM ATP averaged 33% of the particles moved toward the cell body, but imme- (SEM = 4%; N = 6) of the number seen during the 5 min diately inside the plasma membrane a majority of the before the addition of saponin. The frequency of the particles (almost 70%) moved in the anterograde direc- movements declined with time, but usually movement tion (Fig. 3; Table I). The width of the zone below the could still be seen more than an hour after the first axolemma where anterograde movement predominates addition of ATP. However, if the medium was replaced appeared to be less than 1 pm. The mean velocity of by internal medium without ATP, all movement stopped retrograde particle movement (Table I) was the same in within 2 min. Movement could then be reactivated again the interior and at the periphery of the axon; this was by perfusion with medium containing ATP. Movement also true of the mean anterograde velocities (t tests, p > was not reactivated by 1 to 5 mM concentrations of 0.05). The mean retrograde velocity was more than twice adenosine monophosphate or by the nonhydrolyzable as fast as the anterograde velocity (Table I). Mitochon- ATP analogue 5-adenylimidodiphosphate (AMP-PNP). dria also showed more retrograde movement in the center A small number of distinct ATP-dependent movements and more anterograde movement near the surface (Table could be detected with ATP concentrations as low as 10 1282 Forman et al. Vol. 3, No. 6, June 1983

Figure 2. Saltatory movement in a lobster motor axon before and after permeabilization. Panels a to c demonstrate saltatory movement in the interior of a living isolated axon; d to f show the same axon after permeabilization with saponin and demonstrate organelle movement reactivated with ATP. The magnification of each panel is the same; the calibration bar in f = 10 pm. Retrograde movement is to the left. In a to c which were taken at lo-set intervals, one particle (3) moved in the retrograde direction, while a particle (2) and a mitochondrion (I) moved in the anterograde direction. Another mitochondrion (m in a) remained stationary. d to f show the same axon after permeabilization with saponin as described under “Materials and Methods.” Saponin-free medium containing 0.2 mM MgATP was added 1 min before d. d to f show successive lo-set intervals. Two particles (2 and 3) show saltations in the anterograde direction. Between e and f particle 3 moved out of the field of view. The smaller excursion of particle 1 reflects a “tugging” movement (see “Results”). The beaded appearance of the stationary mitochondrion (m in d) is typical of permeabilized axons.

PM. The number and velocity increased with increasing tion) was faster than in the other direction. Typically, ATP concentrations and appeared to be maximal at 1 to this pattern was repeated several times. The proportion 5 rnM. of particles displaying this type of movement usually In addition to long saltations, a second type of organ- increased with time while the frequency of long saltations elIe movement was usually seen after the addition of decreased. We believe that these repeated “tugging” ATP. Particles and mitochondria showed repeated short, movements represent attempts at saltatory movement jerky, back-and-forth movements oriented parallel to the by a trapped particle. The tugging movements required axon. In most cases, the movements were asymmetrical, the presence of ATP and ceased when the ATP was movement in one direction (usually the retrograde direc- removed. The movements reappeared when ATP was The Journal of Neuroscience Vanadate Inhibits Fast Axonal Transport in Permeabilixed Axons 1283

TABLE I Quantitative properties of microscopically visible organelle movements in living lobster motor axons Interior” Periphery’ MeankSEM Mean+SEM Particle velocity bdsec) b Retrograde 1.64 1- 0.05 1.69. + 0.15 Anterograde 0.67 f 0.04 0.59 + 0.02 Percentage of organelles moving in the retrograde direc- tionc,d Particles 81 f 2 31 f 3 Mitochondria 69 f. 4 29 * 3 Frequency of movement’ (organelles/min/lO pm) Particles Retrograde 2.3 f 0.3 0.7 f 0.07 Figure 3. Organelles at the periphery of an axon. The micro- Anterograde 0.5 f 0.04 1.80 f 0.16 scope was focused immediately inside the axolemma. Five par- Mitochondria ticles (small arrows) and one small mitochondrion (large ar- Retrograde 0.8 f 0.1 0.3 f 0.04 rowhead) were moving in the anterograde direction. Bar = 5 Anterograde 0.3 zk 0.04 0.6 + 0.07 pm. “The microscope was focused approximately in the center of the axon (“Interior”) or immediately inside the plasma membrane (“Pe- riphery”). SAPONIN * Velocities of salt&ions were measured from videotape records using 1 I a Colorado Video #610E Video Pointer. The numbers of measurements were: 95 retrograde particles in the interior of 11 axons; 32 retrograde particles at the periphery in 9 axons; 41 anterograde particles in the interior of 11 axons; 44 anterograde particles at the periphery of 7 axons. ’ All organelles crossing a vertical line on the video monitor during a 5-min interval were counted from the videotaped record. Elongated 1OmM ATP organelles more than 1 my” long were counted as mitochondria. In most experiments the vertical line on the screen intersected the equivalent of 25.7 pm of axon, In order to normalize for different magnifications used in a few experiments, the organelle frequencies are expressed as number of organelles/min/lO pm of vertical line. Results for the interior of the axon are the average of 26 counts in 13 axons. Results for the region immediately below the surface are the average of 14 counts in 11 axons. d By averaging the percentage of organelles moving in the retrograde direction from separate counts, an estimate of the variability between -lb ’ b ’ lb 2b 3’0 7’0 counts can be calculated. However, the estimate of the mean is sensitive TIME (minutes) to samples in which only a small number of organelles were counted. Another statistic that gives greater weight to samples where larger Figure 4. Reactivation of saltatory movement with ATP in numbers of organelles were counted is obtained by combining all counts a permeabilixed axon. The number of organelles displaying of the organelles moving in each direction. Calculated this way, the movement (either long saltations or “tugging” movements) percentages of organelles moving in the retrograde direction are: par- during successive 1-min intervals was counted from the video- ticles in the interior 83%; at the periphery 29%; mitochondria in the taped record as described under “Materials and Methods.” interior 71%; and at the periphery 32%. During the 10 min before the addition of saponin, the axon was pre-incubated in internal medium alone. The time when inter- nal medium containing saponin was added was taken as 0. After added again. Similar “tugging” movements of particles 15 min of saponin treatment, movement was reactivated with and mitochondria were often seen in living axons. saponin-free medium containing 5 mM MgATP. This was re- The permeabilization of the plasma membrane by sa- placed 15 min later by 10 InM ATP in order to examine the ponin appeared to be irreversible. Intracellular record- effect of the higher concentration. Movement continued during ings demonstrated that the saponin treatment irrevers- the period from 40 to 65 min (data not shown). ibly depolarized the membrane. When the internal me- dium bathing a saponin-treated axon was replaced with teases (Pant and Gainer, 1980). Identical liquefaction was external medium the axoplasm rapidly liquefied. The seen in Ca2+-free external medium containing 5 mM organelles became completely disorganized and displayed EGTA. On the other hand, if the EGTA in internal Brownian movement in a freely flowing liquid environ- medium was replaced by 5 mu CaCl2 the axoplasm did ment. The structural breakdown is probably due more to not liquefy, but an apparent loosening of structure de- the chaotropic effects of Cl- ions on the axonal cyto- veloped more slowly over a period of 2 to 5 min. Organ- skeleton (Baumgold et al., 1981) than to the action of elles began to show Brownian movement in the radial Ca2+ in depolymerizing microtubules and activating pro- plane while maintaining their longitudinal position in the 1284 Forman et al. Vol. 3, No. 6, June 1983 axoplasm. Mitochondria remained oriented parallel to SAPONIN the long axis. The impression derived from this pattern IA - was that the organelles remained attached to flexible, longitudinally oriented submicroscopic structures but that elements usually cross-linking these structures into a gel had been lost. In spite of this partially disrupted structure, saltatory movements could be reactivated with ATP in the Ca2+-treated axons. Effect of vanadate on fast transport. Vanadate has been used in studies of cell motility because low concen- trations of vanadate ions (in the V+ oxidation state) inhibit dynein but not myosin (see “Discussion”). How- 0 10 i0 4-O ever, vanadate does not readily cross the membranes of living cells (Cande and Wolniak, 1978; Beckerle and B SAPONIN Porter, 1982; Forman, 1982a). We confirmed that an hour I i of incubation in internal or external medium containing 1 mM vanadate did not noticeably affect organelle trans- port in intact axons. Nevertheless, vanadate did inhibit ATP ATP-reactivated saltatory movement in permeabilized axons. After the application of 100 PM sodium orthovan- adate, the number of saltatory movements reactivated by 1 mrvr ATP markedly decreased (Fig. 5A). The long saltations were almost abolished, although some move- 50pM VANADATE ments (mostly low amplitude tugging movements) re- mained even with vanadate concentrations as high as 1 mM. The inhibition was rapidly reversible; movement reappeared when the axon was perfused with vanadate- 0 free medium containing 2.5 mM norepinephrine (Fig. 5A). lb 2-O 3-O 4b The norepinephrine was used because it efficiently re- C verses vanadate inhibition of dynein by reducing the vanadate (Cande and Wolniak, 1978; Gibbons et al., SAPONIN 1978). Norepinephrine alone (2.5 mM) did not stimulate I- ATP-reactivated movement in permeabilized axons that ATP had not been treated with vanadate. The vanadate inhi- bition could also be reversed by washing with vanadate- free medium without norepinephrine. Substantial inhi- bition was reproducibly seen with concentrations of van- adate as low as 50 PM (Fig. 5B). Inhibition was not reliably demonstrable in 25 PM vanadate (Fig. 50. Vanadate has been shown to inhibit the Na+, K+- dependent ATPase (Cantley et al., 1977) that forms the sodium pump of the plasma membrane. To determine i lb 2-O 3-O 4b whether the inhibition of this enzyme by vanadate plays TIME (minutes) any role in the inhibition of saltatory movement in per- Figure 5. Inhibition of reactivated saitatory movement by meabilized axons, we examined the effect of ouabain, vanadate and reversal of the inhibition with norepinephrine another inhibitor of the Na+, K+-ATPase. High concen- (NE). Typical experiments demonstrate the effect oE A, 100 trations of ouabain (0.25 mM) had no effect on the reac- pM; B, 50 pM; and C, 25 pM sodium orthovanadate. The fre- tivation of organelle transport in permeabilized axons. quency of moving organehes was counted as in Figure 4. When ah movement had stopped after 15 min of saponin treatment, Discussion movement was reactivated by the addition of 1 mM MgATP. Microscopic observation of organelle transport in lob- After 5 min, vanadate was added. Fifteen minutes later the ster giant axons. The lobster giant motor axon is partic- vanadate was removed and 2.5 IIIM norepinephrine was added ularly favorable for optical studies of axonal transport. to reduce any residual vanadate. Movement was inhibited by The axons are relatively easy to isolate and display 100 pM and 50 pM vanadate, but 25 pM had little effect. vigorous saltatory movements that continue for several hours in vitro. The glial sheath interferes less with mi- The organelle movements in lobster axons resemble croscopic visualization than does the myelin of vertebrate those previously seen in vertebrate (Smith, 1971, 1972, axons. Lobster axons have frequently been used for phys- 1980; Cooper and Smith, 1974; Forman et al., 1977a, b) iological and neurochemical studies (e.g., Kravitz et al., and crustacean axons (Smith, 1977; Adams, 1982) with 1965; Freeman et al., 1966), but until recently the advan- conventional light microscopy. It has long been recog- tages of these axons for studies of axonal transport have nized that with conventional light microscopy, only the only rarely been exploited (Smith, 1977; Adams, 1982). larger and more refractile organelles were visualized (For- The Journal of Neuroscience Vanadate Inhibits Fast Axonal Transport in Permeabilized Axons 1285 man, 1982c). In vertebrate axons, almost all of the par- move in transport group II. Although transport group I ticles that had been observed were multivesicular bodies, has been extensively studied, very little is known about dense lamellar bodies, and other prelysosomal structures transport group II. Microscopic study of the medium size, (Smith, 1980; Tsukita and Ishikawa, 1980) that move anterogradely moving particles in isolated axons can be mainly in the retrograde direction (Smith, 1971, 1972, used to selectively examine the characteristics of trans- 1977, 1980; Cooper and Smith, 1974; Forman et al., port group II. 1977a). The smaller vesicular and tubulovesicular organ- We have found that in lobster axons, anterogradely elles that are major components of fast anterograde moving particles and mitochondria are both especially transport (Grafstein and Forman, 1980; Smith, 1980; Tsu- concentrated near the axonal plasma membrane. Pre- kita and Ishikawa, 1980) had not been visualized. Re- dominantly anterograde movement near the surface has cently, however, the improved microscopic contrast and also been seen with darkfield optics in other types of resolution provided by the AVEC-DIC technique (Allen crustacean axons (R.S. Smith, personal communication) et al., 1981) has made it possible to detect a population and with AVEC-DIC microscopy in of small vesicles that are rapidly transported in the (Allen et al., 1982b). If anterogradely moving particles anterograde direction (Allen et al., 1982a, b; Brady et al., are also numerous near the axolemma of vertebrate 1982; Breuer et al., 1982). Allen and colleagues report axons, they may not have been detected due to optical that these numerous “small” particles, which apparently interference from the myelin. In fact, there is autoradi- correspond to 30- to 50-nm vesicles and tubulovesicular ographic evidence that anterograde transport is concen- structures (Allen et al., 1982b; Fahim et al., 1982) can be trated close to the plasma membrane of some vertebrate clearly distinguished from a population of “medium” size axons (Lentz, 1972; Byers, 1974). Regional differences in organelles that are larger and more refractile. Although the direction of movement in axons might provide clues small particles are reportedly more numerous in lobster as to the factors that determine the direction of fast axons than medium size particles (Allen et al., 1982a), we transport. were not able to detect structures corresponding to the Organelle transport inpermeabilized axons. With the small particles with our microscopy system, even with an permeabilization technique presented here, giant axons oil immersion objective (numerical aperture = 1.3). Thus, can be used to analyze the biochemical requirements and as in all previous studies that did not use the AVEC-DIC properties of the transport mechanism. Several of our technique, the particles described in this paper are all of results demonstrate that the saponin-treated axons have the “medium” type. The actual size of these “medium” become permeable to small molecules: (1) ATP does not particles cannot be determined by measurement of the cross the plasma membranes of living cells. In our exper- video images. Most of the particles are probably close to iments the very rapid reactivation of movement when or below the limit of resolution of the light microscope. ATP is added, the rapid cessation of movement when the Objects more than an order of magnitude smaller than ATP is withdrawn, and the rapid reappearance of move- the limit of resolution of the light microscope (a measure ment when ATP is again added, all suggest that the of the ability to distinguish two objects as separate) can membrane must be freely permeable to ATP. (2) Vana- still be detected. However, the apparent size of objects at date ions do not readily enter living cells, and incubating or below the resolution limit is inflated by diffraction intact axons for 1 hr in 1 mM vanadate had no effect on (Francon, 1961, pp. 35-58; Allen et al., 1981; Forman, transport. The inhibition of movement by 50 to 100 PM 1982c). Preliminary electron micrographs of lobster axons vanadate and the rapid reversal of the inhibition when reveal clear vesicles with diameters ranging from 0.1 to the vanadate was washed out indicate that the saponin- 0.5 F (K. Lynch, unpublished data) that apparently treated axon had become permeable to vanadate. (3) correspond to the particles that we observed by light Exposure of the permeabilized axons to external medium microscopy. with or without Ca2+, or to internal medium containing Evidence from radiolabeling studies shows that there 5 mM Ca2+, produced rapid changes in axoplasmic struc- are at least two major subcomponents of fast anterograde ture. Nonpermeabilized axons, on the other hand, func- transport (Lorentz and Willard, 1978; Levine and Willard, tion in these same media for many hours without mor- 1980; see Grafstein and Forman, 1980). The fastest (trans- phological changes. Whether there is an effective upper port group I) has been most frequently studied and limit on the size of molecules that can freely enter the probably carries synaptic vesicle and plasma membrane saponin-treated axons remains to be determined. precursors (Grafstein and Forman, 1980). The slower Adams (1982) has recently described a method for component (transport group II) includes mitochondria reactivating particle transport in giant axons from Car- (Lorentz and Willard, 1978) and nonmitochondrial ma- cinus maenas permeabilized with high voltage dis- terials (Levine and Willard, 1980). The smallest particles charges. Although the results reported by Adams are in lobster axons, those that are detectable only with generally similar to our findings in lobster axons, there AVEC-DIC microscopy, have been reported to move in are differences in details between the two studies: (1) the anterograde direction with a mean velocity of 3.8 Apparently Adams did not detect movement with ATP m/set (Allen et al., 1982a). These would be expected to concentrations below 500 PM, whereas we detected slight form transport group I. The “medium” particles that we movement at 10 PM. It may be that we were able to observed moving in the anterograde direction had a mean detect more moving particles due to the sensitivity pro- velocity of 0.6 to 0.7 pm/set, which is about the same as vided by Nomarski optics and video time-lapse recording. that of the anterogradely moving mitochondria. There- (2) Adams reported obtaining inhibition of movement fore, these particles, along with the mitochondria, must with vanadate concentrations as low as 5 PM, whereas we 1286 Forman et al. Vol. 3, No. 6, June 1983 could not reliably distinguish inhibition below 50 PM. is still possible that an unidentified, vanadate-sensitive Adams’ experiments with vanadate are not described in protein, different from dynein, is involved in fast axonal detail, and we are unable to explain this discrepancy. (3) transport. Adams reported that 0.5 to 1 mM Ca2+ causes rapid, Vanadate also inhibits saltatory organelle movement protease-dependent dissolution of the axoplasm; we ob- in several types of permeabilized non-neural cells (For- served a slower, partial disruption of axoplasmic struc- man, 1981, 1982a, b; Stearns and Boggs, 1981; Beckerle ture. On the whole, however, the degree of agreement and Porter, 1982; Clark and Rosenbaum, 1982; Stearns between the results of these two independent studies is and Ochs, 1982). There have been two reports that van- remarkable. adate fails to block the fast anterograde transport of Inhibition of fast organelle transport by vanadate. radioactive proteins when injected into the cell body Vanadate anions in the V+ oxidation state inhibit a (Isenberg et al., 1980) or axon (Goldberg, 1982) of inver- variety of ATPases and other enzymes (Cantley et al., tebrate . However, it is possible that the intra- 1977; Gibbons et al., 1978; Goodno, 1979). Vanadate has cellular vanadate concentration achieved in those exper- been used as a probe in studies of cell motility because iments was not high enough to inhibit cytoplasmic dy- dynein and myosin, the major known mechanochemical neins (see Forman, 1982a). Furthermore, some cells can coupling ATPases, differ markedly in their sensitivity to overcome the inhibitory effect of microinjected vanadate vanadate. Low concentrations of vanadate (0.1 to 10 PM) within 10 to 20 min after injection, probably by reducing rapidly inhibit isolated flagellar dynein and the dynein- the vanadate (Stommel et al., 1980). A brief inhibition dependent beating of cilia and flagella (Cande and Wol- by vanadate would not have been detected with the niak, 1978; Gibbons et al., 1978; Kobayashi et al., 1978; labeling methods. Combining microinjection into living Hisanaga and Sakai, 1980; Warner and Mitchell, 1980; isolated giant axons with direct microscopic observation Shimizu, 1981). This inhibition can be rapidly reversed of the effects might help to resolve some of the discrep- by removal of the vanadate or by its reduction with ancies between the results of microinjection and permea- reagents such as norepinephrine (Gibbons et al., 1978; bilization experiments. Kobayashi et al., 1978; Warner and Mitchell, 1980). On Light microscope observation of intra-axonal organelle the other hand, vanadate does not significantly inhibit movement provides information about fast axonal trans- purified myosin except at higher concentrations (500 to port with much greater spatial and temporal resolution 1000 a~) (Cande and Wolniak, 1978; Gibbons et al., 1978; than can be obtained with other techniques. However, Kobayashi et al., 1978; Goodno, 1979). The inhibition of microscopic analysis of transport in vertebrate axons has myosin by vanadate requires several hours to develop been hindered by the difficulty of isolating single axons, and is then irreversible (Goodno, 1979). Furthermore, the delicacy of the axons, and optical interference from vanadate does not inhibit well established myosin-based the myelin sheath. Large motor axons from lobster walk- motility such as myofibril contraction (Cande and Wol- ing legs are relatively easy to isolate and survive well in niak, 1978) or cytokinesis (Cande et al., 1981) in permea- vitro. Saltatory movements of particles and mitochondria bilized cells. Therefore, rapid, reversible inhibition of a are easily visualized in these axons. The permeabilization motile system by low concentrations of vanadate is con- protocol presented here makes it possible to study the sistent with a dynein-based mechanism, rather than one mechanism of transport with probes that cannot enter involving myosin (Cande and Wolniak, 1978; Kobayashi living axons. The regional difference in the direction of et al., 1978). The few cytoplasmic (Hisanaga and Sakai, organelle movement may be useful for studying the fac- 1980; Pratt et al., 1980) or membrane-associated (Dentler tors that determine the direction of movement. Thus, et al., 1980) dyneins (or dynein-like molecules) that have isolated giant axons appear to be highly favorable for been studied have been found to be less sensitive to microscopic studies of the basic mechanisms underlying vanadate than dyneins from ciliary and flagellar axo- fast axonal transport. nemes. Nevertheless, the nonaxonemal dynein-like References ATPases are reversibly inhibited by vanadate in the Abercrombie, R. F., L. M. Masukawa, R. A. Sjodin, and D. R. range of 10 to 100 PM, concentrations lower than those Livengood (1981) Uptake and release of 45Ca by My&& needed to inhibit myosin. The sensitivity of dyneins to axoplasm. J. Gen. Physiol. 78: 413-429. inhibition by vanadate decreases in conditions of high Adams, R. J. (1982) Organelle movement in axons depends on ionic strength (Hisanaga and Sakai, 1980). The high ionic ATP. Nature 297: 327-329. strength of the medium used for permeabilization may Allen, R. D., N. S. Allen, and J. L. Travis (1981) Video-enhanced have contributed to our inability to detect inhibition at contrast, differential interference contrast (AVEC-DIC) mi- vanadate concentrations lower than 50 pM. Thus, the croscopy: A new method capable of analyzing microtubule- finding that 50 to 100 PM vanadate rapidly and reversibly related motility in the reticulopodial network of Allogromia inhibits organelle movement in permeabilized axons sug- laticollaris. Cell Motil. 1: 291-302. gests that fast axonal transport may involve a dynein- Allen, R. D., R. J. Lasek, S. P. Gilbert, A. J. Hodge, and C. K. like molecule. The nucleotide specificity of the reacti- Govind (1982a) Fast axonal transport in lobster axons. Biol. Bull. 163: 379-380. vated movements (Forman et al., 1982b) is also consistent Allen, R. D., J. Metuzals, I. Tasaki, S. T. Brady, and S. P. with a dynein-based mechanism. Because dyneins inter- Gilbert (1982b) Fast axonal transport in squid giant axon. act with microtubules, a dynein-based mechanism would Science 218: 1127-1129. be consistent with the considerable body of evidence that Baumgold, J., S. Terakawa, K. Iwasa, and H. Gainer (1981) microtubules are essential for fast axonal transport (re- Membrane-associated cytoskeletal proteins in squid giant viewed in Grafstein and For-man, 1980). Nevertheless, it axons. J. Neurochem. 36: 759-764. The Journal of Neuroscience Vanadate Inhibits Fast Axonal Transport in Permeabilized Axons 1287

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